Integrated approach to the selection of new probiotics for human application

Scuola di Dottorato per il Sistema Agro-alimentare

Doctoral School on the Agro-Food System ciclo XXVIII

S.S.D: AGR/16, BIO/19, AGR/15

Integrated approach to the selection of new probiotics for human application

Coordinator: Ch.mo Prof. Antonio Albanese

Tutor: Prof. Maria Luisa Callegari

Dott.ssa Marina Elli

Candidate: Elena Guidesi

Matriculation n.: 4110923

Academic Year 2014/2015

I

INDEX

CHAPTER 1

GENERAL INTRODUCTION

1. Probiotics 1

2. Human microbiota 2

3. Lactic acid bacteria 4

3.1 Genus Lactobacillus 5 4. Genus Bifidobacterium 6 5. “Non-conventional” probiotics 8 6. Probiotics: guidelines for use in foods and food supplements 9 6.1 Amount of microorganisms 9 6.2 Safety of probiotics 9 7. Selection and characterization of Lactobacillus and Bifidobacterium strains: “conventional screening” 11

7.1 Identification and typing 12

7.2 Antibiotic sensitivity 14

7.3 Survival to GIT stressing conditions 14

8. Targeted screening focused on the application 15

8.1 Prevention of pathogen adherence 16

8.2 Prevention of diarrhea and intestinal infections 17

8.3 Stimulation of the immune system and beneficial effects on allergic reactions 18

9. Gut-brain axis 21

10. Market of probiotics 23

11. References 24

Aim of the thesis CHAPTER 2 SELECTION OF PROBIOTIC CANDIDATES FOLLOWING CONVENTIONAL IN-VITRO CRITERIA

2. Materials and methods 33

2.1 Isolation of LAB 33

2.2 DNA extraction 33

2.3 Identification of isolated strains 34

2.4 Typing by rep-PCR 34

2.5 Evaluation of resistance to gastric juice 35

2.6 Evaluation of resistance to bile salts 36

2.7 Antibiotic susceptibility evaluation 36

3. Results 37

3.1 Identification of isolated strains 37

3.2 Effects of bile salts on viability 46

3.3 Effects of gastric acidity on viability 50

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3.4 Antibiotic susceptibility testing 50

4. Conclusions 53

5. References 54

CHAPTER 3 SPRAY-DRYING ENCAPSULATION OF PROBIOTICS FOR ICE-CREAM APPLICATION

2. Materials and methods 61

2.1 Preparation of modified ice-cream base formulations 61

2.2 Preparation of probiotics enriched formulations 62

2.3 Ice-cream preparation 62

2.4 Statistics 63

3. Results and discussion 64

3.1 Preparation of modified ice-cream base formulations 64

3.2 Preparation of probiotics enriched formulations 65

3.3 Ice-cream preparation 67

4. Conclusions 67

5. References 68

CHAPTER 4 UPDATING THE CRITERIA FOR PROBIOTIC SELECTION AND CHARACTERIZATION FOR HUMAN USE

1.1 Persistence and survival in the human gut 72

1.2 Safety for the use in human nutrition 72

1.3 Colonization ability 73

1.4 Antimicrobial potential 73

1.5 Immunomodulatory actions 74

1.6 In-vitro models of cardiovascular diseases 74

1.7 In-vitro anti-cancer models 75

2. Development of innovative screening platforms 75

3. References 78

CHAPTER 5 LACTOBACILLI AND BIFIDOBACTERIA PROBIOTICS AMELIORATE EXPERIMENTAL AUTOIMMUNE MYASTHENIA GRAVIS AND ENCEPHALOMYELITIS

2. Results 85

3. Discussion 89

4. Materials and methods 92

5. References 99

III

CHAPTER 6

NEW PROBIOTIC CANDIDATES VS BIOGENIC AMINES: IN-VITRO SCREENING CRITERIA

1. Introduction 118

2. Materials and methods 120

2.1 Strains and culture conditions 120

2.2 DNA extraction and PCR primers and procedure 121

2.3 Protein extraction from probiotic cells 121

2.4 Quantification of the extracted proteins 122

2.5 Enzimatic assay for diamine oxidase 122

3. Results 122

3.1 BA synthesis by novel probiotic lactobacilli 122 3.2 BA degradation by novel probiotic lactobacilli 123

4. Conclusions 123

5. References 130

CHAPTER 7

SUMMARY AND CONCLUDING REMARKS

Supplementary material Acknowledgments

Chapter 1: General introduction

1

Probiotics

The definitions given to the term probiotic are numerous. The FAO (Food and Agriculture Organization) and WHO (World Health Organization) define probiotics living organisms which, when administered in adequate amounts, bring benefits to the health of the host. The term probiotic is therefore closely related to the term health: the human intestinal bacterial flora can in fact be easily weakened by different factors such as administration of drugs and stress situations and for this reason it’s important to consider a way to support and strengthen the same. Here come into play foods containing probiotics, in line with the definition given above, they are in fact able to optimize the intestinal flora in order to achieve the well-being and a good health.

The International Scientific Association for Probiotics and Prebiotics (ISAPP) organized on 23 October 2013 a meeting of experts on probiotics to revise the definition of the term “probiotic”

and to develop clear guidelines for a more conscious use of the term. After the meeting, individual panelists have written a summary document that was approved by all the members before submission called precisely “consensus statements”. The panelists established that the FAO/WHO definition of the term “probiotic” remains relevant to validate a new probiotic strain with one modification. A specific strain can be considered as probiotic even if randomized controlled trials are not conducted on it, if the strain belongs to species for which exists a scientific evidence about their beneficial effects on human health. Furthermore, the panel did not agree with the fact of require investigational new drug applications for probiotics food because this activity would increase the costs of research and it is very difficult for products that do not respect pharmaceutical standards. Finally, the panel considers that a strong evidence about the beneficial effect of the probiotic is necessary both at strain-specific or group level and stresses the need to improve communication to the public on the beneficial effects of probiotics.

Our digestive system contains hundreds of viable organisms. There are more than 400 species bacteria that live in the gastrointestinal tract, constituting a real ecosystem. The health of the gastrointestinal flora is not only essential for the correct functioning of the bowel, but it is also important to increment the body's natural defenses against invading bacteria and pathogens.

2 So food containing probiotics, or friendly bacteria, help to sustain the vitality of our natural defenses.

Human microbiota

The human body is inhabited by a large number of bacteria, viruses and other unicellular eukaryotic organisms. The set of micro-organisms that live in peaceful coexistence with their human host is defined "microbiota" or "normal microflora". The human microbiota consists of a biomass really huge, there are not less than 10^14 bacterial cells.

The microbiota is an intestinal ecosystem formed by a large number of ecological niches, which are the house of the bacterial population consisting of numerous species and high number of strains. It is closely related with the intestinal mucosa, or with the epithelial interface, that it is, after the respiratory, the largest surface of the body, being approximately 250 - 400 m^2.

Microbial colonization is a process that begins at birth, when the baby comes into contact with bacteria from the urogenital tract of the mother. Subsequently there is the development of many other microbial species, leading to the establishment of complex interactions between the bacteria themselves and between the latter and the human organism. The microbiota is influenced by many factors such as the composition of the diet of the subject, the body temperature, the use of drugs, the quantity of ingested food and other physiological characteristics; accordingly, it is subject to variations in relation to the changes that occur in the life of an individual.

Man born germ-free. During the first year the intestinal stretch of the baby passes by a condition of sterility to a colonization very dense. Breast milk is an important source of bacteria that influences the development of the intestinal microbiota of the infant. Lactic acid bacteria, bifidobacteria, streptococci and staphylococci are the main groups represented and, between these, the first two are those that will operate positively in different periods of human life. A two-years life of the new born intestinal microflora is almost stabilized.

Any portion of the gastrointestinal tract is colonized by specific bacteria that adapt to local conditions.

The oral cavity due to its characteristics of temperature, pH and nutrient availability is a favorable environment for the growth of microorganisms. The main bacterial genera present in

3 the oral cavity are Streptococcus and Lactobacillus and also Actinomyces are quite abundant in this cavity; however, there is a considerable variation of the microflora in the case of plate or of oral infections.

In the mouth, in addition to the bacteria, are also commonly present Archea, yeasts, especially of the genus Candida, and other microorganisms such as mycoplasma and protozoa.

The environment of the stomach has a low pH (less than three), which makes difficult the survival and growth of microorganisms. The gastric flora is quantitatively very poor, so it is possible to define the content of the stomach almost sterile. Only some bacteria are able to multiply in this section of the digestive system; they are acid tolerant and they consist mainly of lactobacilli (Lactobacillus acidophilus and L. plantarum) and streptococci.

The number of bacterial cells present in the gastrointestinal tract of a mammal shows a continuum increasing, varying from 10^3 bacteria/g in the stomach and duodenum, to 10^4- 10^7 in jejunum and ileum, to over 10^12 cells/g in the cecum and in the colon (Figure 1). The acid environment of the stomach has a negative impact on the most bacteria that pass through it, so this is the first defensive barrier to contamination from the outside. Most of the bacteria resides in the lower part of the digestive system, especially in the large intestine, also because in the most proximal tract bile and pancreatic secretions are toxic or not favorable for the growth of the most microorganisms.

Figure 1: Distribution of intestinal microflora (Customprobiotics.com)

4 This microflora normally is not pathogenic and helps to maintain the state of health of the host, facilitating the absorption of nutrients, degrading substances potentially harmful or allergenic proteins and generating immune responses such as to prevent inflammation in the intestine (Chin et al., 2000).

It was seen that among the components of the gut flora, lactic acid bacteria, such as bifidobacteria and lactobacilli, are able to exert benefits for the health of the host. Lactobacilli are ubiquitous inhabitants of our body being found in numerous districts: gastric, intestinal, oral, urogenital, etc. They are also an important component of the intestinal microbiota, not so much in quantitatively terms but mainly from the functional point of view, and because they are used in probiotic applications. The intake of lactobacilli as probiotics is aimed to maintain the intestinal microbial flora constantly in balance in order to avoid situations of intestinal dysbiosis, characterized by an alteration of the same with the predominance of pathogenic bacteria.

Lactic acid bacteria

Lactic acid bacteria constitute a large and diverse family of microorganisms that, from the fermentation of sugars, mainly produce lactic acid. These bacteria, essentially ubiquitous, are normally present in food products and are widely used at industrial level because they intervene in many fermentation processes. In particular, they are found in foods and beverages made with plant materials such as sauerkraut, pickles, silage, fodder and beer; they are also agents of the cheese ripening and, together with yeast, are involved in the leavening of bakery products. Some are part of the normal microbiota of the animals and can be ingested by humans as well as probiotics. These microorganisms may have the form of coco or rod, are Gram positive, catalase negative, unable to move, they don’t produce spores, and anaerobic- microaerophilic, in fact they well multiply at low oxygen concentrations. In addition to not possess the catalase they don’t even have the enzymes nitrate reductase and cytochrome oxidase; in fact, they don’t have a respiratory chain but they have a fermentative metabolism.

They are heterotrophic microorganisms, adapted to live on complex substrates and which, as a source of energy, require not only carbohydrates but also vitamins, nucleotides, mineral elements such as manganese and magnesium (used as cofactors of metabolism) and amino

5 acids. They are in fact very demanding from the nutritional point of view and they require particular regards for nitrogen and vitamin sources while they are being diversified for the carbonaceous sources. Among lactic acid bacteria there are mesophilic and thermophilic species; the optimum pH for growth varies from 7 to 5, otherwise they well tolerate high acidity, some species are in fact able to grow at pH values very low, approximately of three units. This taxa, belonging to the phylum Firmicutes, class Bacilli and order Lactobacillales, includes a variety of genus such as Abiotrophia, Aerococcus, Carnobacterium, Enterococcus Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus and Weissella.

Among them the genus Lactococcus, Streptococcus and Lactobacillus are particularly important (Makarova and Koonin, 2007).

Genus Lactobacillus

Lactobacillus is a genus of Gram positive and rod-shape bacteria belonging to the family of Lactobacillaceae. The genus Lactobacillus is widely distributed in nature (there are over 100 species) although, like all lactic acid bacteria, it has high nutritional requirements. Lactobacilli can grow in a temperature range between 5°C and 53°C with optimal values of 30 - 40°C. They have an optimum pH for growth of 5.5 to 5.8 but they can still grow even at pH less than 5.

These bacteria produce mainly lactic acid by fermentation of sugars but also acetic acid, ethanol, carbon dioxide and other secondary compounds. The production of lactic acid leads to a reduction of the pH of the medium in which they grow, and this acidification of the environment is able to inhibit the growth of certain pathogenic microorganisms. On the basis of how they use glucose during fermentation process they may be divided into homofermentative (if they produce more than 85% of lactic acid from glucose) and heterofermentative, responsible in this case of heterolactic fermentation that products for about 50% lactic acid and for about 50% other substances (they produce lactic acid, carbon dioxide, ethanol and / or acetic acid in equimolar amounts). In relation to the metabolism, the species of Lactobacillus can be divided into three groups:

• Obliged homofermentative: include microorganisms that ferment hexoses carbohydrates with production of only lactic acid, they are not able to ferment pentose and they don’t produce gas.

They are part of this group the species L. delbrueckii, L. acidophilus, L. helveticus and L.

salivarius.

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• Facultative heterofermentative: include lactobacilli which produce lactic acid by fermentation of hexoses but some species, in certain conditions, also produce acetic acid, formic acid and / or ethanol. In addition, they also ferment pentoses with the production of lactic acid and acetic acid. This group includes the species L. casei, L. curvatus, L. plantarum, L. sakei.

• Obliged heterofermentative: they ferment hexoses with formation of lactic acid, carbon dioxide, acetic acid and / or ethyl alcohol. They are also able to ferment pentose producing lactic acid and acetic acid. They are included in this group the species L. fermentum, L. brevis, L.

collenoides, L. hilgardii, L. fructivorans.

Lactobacilli are present in different habitats, in addition to the digestive and urogenital human system other habitats are represented by silage, soil, water, cereals and fermented foods (milk, meat and vegetables).

The different species of lactobacilli have adapted differently: they have developed different characteristics associated with the environment in which they live. The broad ecological distribution of lactobacilli makes them an interesting subject of research on genome evolution and lifestyle adaptation. To explore evolutionary mechanisms that determine genomic diversity of different species of lactobacilli, nowadays it is used the comparative analysis of genomes.

The analysis of the genome have contributed to a more complete understanding of their phylogenetic position and revealed important details about their specialized adaptation to a specific environment. Van de Guchte et al., 2006, through genome sequencing, have demonstrated the adaptation of L. bulgaricus to the milk environment through the protocooperation with Streptococcus thermophilus and these species are in fact the species most used for yogurt production. Also Cremonesi et al., 2013 through the genomic analysis explained the widespread use of L. helveticus in cheese technology. About that it has revealed genes responsible for key metabolic functions such as proteolysis, lipolysis, and cell lysis and these genes can facilitate the production of cheese and cheese derivatives. These studies provide insights about mechanisms for genome evolution and lifestyle adaptation of these ecologically flexible and industrially important lactic acid bacteria.

Genus Bifidobacterium

Microorganisms of the genus Bifidobacterium are non spore-forming, non motile and they can show various shapes, which the most typical ones are slightly bifurcated club-shaped or

7 spatulated extremities. They are strictly anaerobic, although some species can tolerate low oxygen concentrations and they have a fermentative metabolism. Tissier described these bacteria at the beginning of the twentieth century (Tissier, 1900). They were first included among the family Lactobacillaceae, but in 1924 the species L. bifidum was reclassified into the new genus Bifidobacterium.

The species of the genus Bifidobacterium form a coherent phylogenetic group and show over 93% similarity to the 16S rRNA sequences among them (Satokari et al., 2003). This genus is included in the phylum Actinobacteria, class Actinobacteria, subclass Actinobacteridae, order Bifidobacteriales, family Bifidobacteriaceae, and it includes Gram-positive bacteria with high content of G+C.

All the currently known Bifidobacterium isolates are from a very limited number of habitats, that is human and animal GITs, food, insect intestine, and sewage (Felis and Dellaglio, 2007;

Ventura et al., 2004). Strains most commonly found in human intestines and feces are those belonging to the species Bifidobacterium catenulatum, B. pseudocatenulatum, B. adolescentis, B. longum, B. breve, B. angulatum, B. bifidum and B. dentium, and the typical species isolated from functional foods is B. animalis subsp. lactis (Masco et al., 2005). Often in commercial products are present species other than those typical of the human gut. An example is given by B. lactis, a species widely used in commercial products thanks to its greater capacity for survival and resistance to stress.

The genome structure of this group of microorganisms remains largely unexplored; Milani et al., 2014 sequenced the genomes of 42 subspecies belonging to the genus Bifidobacterium and they used this information to explore the genetic picture of this bacterial group. They suggest that the evolution of this genus was substantially influenced by genetic adaptations to obtain access to glycans, indicating in this way that this mechanism represents a strong evolutionary force in shaping bifidobacterial genomes.

Another application of the analysis of the genome revealed the genetic elements responsible for the use of carbohydrates by bifidobacteria of the intestinal microbiota. Particularly, Milani et al., 2015 have performed the analysis of the genome of 47 strains of bifidobacteria showing that, in this bacterial genus, the genes for the catabolism and degradation of carbohydrates are better represented compared to those found in other bacteria of the intestinal microbiota and this ability to degrade glycans reflects the availability of carbon sources of the human gut.

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“Non-conventional” probiotics

In recent years new species of bacteria with probiotic properties have been discovered. Among them there is the species Faecalibacterium prausnitzii, an important commensal bacterium of the human gut flora. In healthy adults, the species F. prausnitzii represents more than 5% of the bacteria in the intestine, making it one of the most common gut bacteria. In the last few years an increasing number of studies have described the importance of this commensal bacterium as a component of the healthy human microbiota demonstrating that lower than usual levels of F. prausnitzii in the human gut have been associated to dysbiosis in several human disorders (Miquel et al., 2013). Particularly, it was shown that a decrease in the levels of this bacterial species is closely related to Crohn's Disease as demonstrated by Wright et al., 2015. They studied the taxonomic shifts in patients with this inflammatory bowel disease revealing significant changes in microbial composition: the abundance of Bacteroidetes is increased and Firmicutes decreased in Crohn's disease compared with healthy controls. Enterobacteriaceae, specifically Escherichia coli, is enriched while F. prausnitzii is found at lower abundance.

In order to develop a novel probiotic, several strains of this bacterial species were isolated and characterized by Foditsch et al., 2014 showing that the concentrations of the short chain fatty acids acetate, butyrate, propionate and isobutyrate in the culture media change in the presence of bacterial growth. They observed a significative reduction in the concentration of acetate followed by a concomitant increase in the concentration of butyrate, suggesting that the isolates are able to consume acetate present in the media and producing butyrate. This is a positive effect because butyrate has many benefits to the colonic epithelial cells and so the selection of strains that produce higher amounts of butyrate is extremely important for the development of a new potential probiotic.

Another “non-conventional” probiotic is represented by the species Akkermansia muciniphila, a species of the new genus, Akkermansia, proposed in 2004 by Derrien et al. A. muciniphila is a Gram-negative, strictly anaerobic, non-motile, non-spore-forming and oval-shaped. It is able to use mucin as its sole source of carbon and nitrogen, and able to colonize the gastrointestinal tracts of human. Extensive research is being undertaken to understand its association particularly with inflammation (Everard et al., 2013). Infact, A. muciniphila is believed to have anti-inflammatory effects in humans, and studies have shown inverse relationships between A.

muciniphila colonization and inflammatory conditions such as appendicitis or irritable bowel

9 syndrome. In one study, reduced levels of A. muciniphila are correlated with increased severity of appendicitis. In a separate study, patients with irritable bowel syndrome were found to have lower levels of A. muciniphila in their intestinal tract than individuals without this syndrome (van Passel et al., 2011).

Probiotics: guidelines for use in foods and food supplements

In order to protect the health of consumers, foods containing probiotics are subject of attention, as confirmed by the elaboration of international guidelines aimed to safeguard the quality of the products.

Ministerial guidelines (revision of 2013) define first the requirements required to use microorganisms as probiotics. They must in fact be sure to use in humans and to be alive and vital in such quantity as to allow multiplication and their activity in the intestine (for reasons that are explained after).

Amount of microorganisms

Ministerial guidelines specify that the minimum quantity of microorganisms able to temporarily colonize the intestine is 10^9 living cells for strain and for day. The recommended daily dose of the product must therefore possess a charge in viable cells equal to 10^9 for at least one of the strains present in the product. This is a topic of much debate because despite regulatory orientation is the one just mentioned, some leaders of the sector say that in a mixed product all species must have a charge of at least 10^9.

Safety of probiotics

To ensure the safety of the microorganism used, it’s necessary an identification of the same on the strain level. First of all, for safety and efficacy reasons it is important that each strain under study is clearly identified, in fact different bacterial strains belonging to the same species may play different beneficial actions in the host. The species can be identified by determination and analysis of the DNA sequence coding for the 16S rRNA or through the hybridization of nucleic acids, the strain can be characterized by PFGE (Pulse Field Gel Electrophoresis). Some of these methods are technically obsolete, however, they are still required from regulatory point of

10 view. Some of these techniques are no longer used because they are time-consuming and not cost efficient but they are highly reliable because they allow to obtain highly reproducible profiles unlike other new methods.

The clear identification of strains and their amount in the products are nowadays subject to careful regulation given the frequent cases of misidentification and mislabeling that occurring.

An example is shown by the control made by Aureli et al., 2010 who tested 72 of probiotic food supplements produced and distributed on the Italian market during 2005-2006. They have shown that 87% of the products analyzed do not comply with the guidelines and the differences were both quantitative and qualitative (number, types and viability of microorganisms).

Moreover, even though most labelled supplements indicate the presence of Bifidobacterium bifidum, this organism was detected only rarely and always as dead cells. This study is a clear demonstration that on the Italian market, there are commercial probiotic products that do not correspond to what is written on the label and consequently to what the Italian guidelines suggest.

In order to consider safe a bacterial strain is necessary a reliable taxonomical identification as described above, and the evaluation of the sensitivity to antibiotics. As is known, in fact, the phenomenon of bacterial resistance to antibiotics is often based on the presence in the bacterial cells of mobile genetic elements, such as plasmids and transposons, which can be transmitted from one organism to another promoting the horizontal spread of resistance.

Particularly the intrinsic resistance to antibiotics is not a problem, the problem arises when the resistance determinants may be transferred to other bacterial strains; evaluation of antibiotic sensitivity is necessary to ensure the absence of acquired or potentially transmissible resistances. For the evaluation of the bacterial safety EFSA (European Food Safety Authority) has introduced the concept of QPS ("Qualified Presumption of Safety"), in practice it was developed a list of bacterial groups suitable for QPS assessment: for them it is not necessary safety assessment but only the determination of sensitivity to antibiotics (EFSA, 2013).

It is on the basis of this regulation and the international community on probiotics that researchers should be directed to develop products really useful to the consumer, taking into account that different types of consumers need different probiotics: probiotics are in fact

11 influenced by various factors such as age of the host, his physiological / pathological conditions and his diet. It is also necessary to increase the effectiveness of products containing probiotics and, for that, it is important to pay attention in each phase of the production: from the selection of bacterial strains to the classification and identification of the same and then proceed to the safety assessment and the determination of the functional characteristics of the strains.

Selection and characterization of Lactobacillus and Bifidobacterium strains:

“conventional screening”

A pool of bacterial strains is initially characterized from the taxonomical point of view and for safety for use in human (“conventional screening”) and then only bacterial strains that possess these features of safety were subjected to a more specific screening, depending on the application to which they will be destined (targeted screening application-focused).

They will therefore be intended to a process of "selection funnel" aimed at the realization of a series of in vitro tests to assess their applicability as probiotics for the promotion of human health.

The most common bacteria used as probiotics belong to the genus Lactobacillus and Bifidobacterium. The selection process of a strain to be used as a probiotic is based on a multi- step approach:

According to FAO/WHO guidelines it is necessary to identify the microorganism to species/strain level because probiotic effects are strain specific (FAO/WHO, 2006). Further characterization of strains is important especially for safety assessment, in fact in order to guarantee fermented products and food supplements that are safe for consumption, some characteristics of the novel lactobacilli and bifidobacteria strains must be studied to ensure their safety.

Within the “Conventional screening” several in vitro tests are conducted for a further characterization of strains to ensure that the bacteria are able to reach the intestine alive and vital, an important requirement to ensure their effectiveness. In Figure 1 are represented the main phases of the “conventional screening”.

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Figure 1: Main phases for identification and characterization of probiotics (Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food London, Ontario, Canada, 30 April – 1 May 2002)

It is well accepted that an effective human probiotic should be of human origin and this is due to the fact that human intestine is different from those of animals, so strains isolated from animal intestine may not be suitable for humans (O’Sullivan, 2001).

For the isolation of new strains, classical cultivation techniques must be employed, particularly selective media and specific culture conditions are used for the isolation of strains from human fecal samples (Delgado et al., 2006).

Identification and typing

In the last decade molecular methods, mainly based on the analysis of nucleic acids by using polymerase chain reaction (PCR) amplification, have been developed for identifying probiotics (Ben-Amor et al., 2007).

The study of ribosomal RNA genes is the most common method for the identification of bifidobacteria and lactobacilli. Bacterial ribosomes are composed by proteins and three

13 ribonucleic acids: 5S RNA, 16S RNA, and 23S RNA. The analysis of the 16S rDNA allows to discriminate all Bifidobacterium and Lactobacillus species and their respective subspecies and biotypes (Germond et al., 2002; Matsuki et al., 2003; Ventura et al., 2006) by sequencing fragments of DNA previously amplified by “universal” or group-specific primers. In addition to provide a precise identification, comparison of the 16S rRNA gene sequences allows to understand the evolutionary relationships among the distinct species. The analysis of 16S rDNA sequences has shown that the classification of lactobacilli in three metabolic groups (obliged homofermentative, facultative heterofermentative, obliged heterofermentative) is not in accordance with their evolutionary relationships. In fact Lactobacillus species can be grouped into several groups and don’t form a coherent phylogenetic unit (Felis and Dellaglio, 2007;

Satokari et al., 2003).

Methods based on the PCR are widely used and allow the differentiation between strains of the same species, by examining patterns generated by amplification of DNA fragments these methods offer a potential for probiotic strain typing. Particularly, REP (repetitive extrogenic palindromic) PCR examine specific patterns of repetitive DNA elements. REP sequences are short DNA fragments detected in the extragenic space, and they are dispersed in the bacterial genomes (Ventura et al., 2003; Tobes and Ramos, 2005).

Recently, more robust typing methods have been applied to species and strains of Lactobacillus and Bifidobacterium such as the multilocus sequence typing (MLST) scheme. This method made use of an automated DNA sequencing procedure to characterize the alleles present at different housekeeping gene loci. As it is based on nucleotide sequences, it is highly discriminatory and it provides unambiguous results. This powerful technique has been applied for phylogenetic studies of strains of Lactobacillus and a variant of MLST, called multilocus variable-number tandem repeats analysis (MLVA) was used for the subtyping of L. casei / L. paracasei.

Also the genome sequence analysis is nowadays used as a new taxonomic technical approach and it provides insights on bacterial evolution thus influencing bacterial taxonomy. Genomic sequence information has in fact been proposed for defining a new genomic-phylogenetic species concept for prokaryotes. For example, comparative genomics has supported the idea that the lactobacilli don’t form a coherent phylogenetic group but it seems that some species (L. salivarius, L. plantarum) are more closely related to Enterococcus faecalis than to other lactobacilli. An example of genomic techniques is the comparative genome hybridization (CGH)

14 that can be used to determine the genome content of a bacterial strain for which is not known the genome sequence.

Antibiotic sensitivity

Lactic acid bacteria are naturally resistant to many antibiotics, but in most cases the resistance is not transferable by horizontal gene transfer (HGT). However when the antibiotic resistance is plasmid-associated it can be transferred to other species and genera, so it is important to select strains lacking the potential to transfer genetic determinants of antibiotic resistance. About that, Mathur and Singh observed that LAB used in probiotics may be a source antibiotic- resistant genes which could be potentially transferred to pathogenic bacteria (Mathur and Singh, 2005). Therefore they suggested systematic screening for antibiotic resistance in probiotic strains: In the case of presence of antibiotic resistance the approach to follow is suggested by Courvalin, 2006, consisting in the identification of the resistance gene, evaluation of the ability to transfer the resistance, characterization of the biochemical mechanism of resistance and elucidation of the genetic basis for resistance. If the resistance feature is not associated with a mobile genetic element, the risk of transfer of resistance would be assessed as low.

The experimental methods used to test the sensitivity to antibiotics are described in Chapter 2.

Survival to GIT stressing conditions

The ability to survive to stressful gastrointestinal tract (GIT) conditions (low pH and high bile salts concentrations) is an important criteria used for the selection of probiotics strains. The transit of probiotics present in food through the GIT takes variable times and is submitted to different stressful conditions. After mastication, the first barrier that bacteria must overcome is the low pH of the stomach with values ranging from 1 to 3. Into the duodenum the pH value rises to 6-6.5 but bile salts reach concentrations ranging from 1.5 to 2% during the first hour of the digestion (Noriega et al., 2004). For the screening of probiotics so it’s important to simulate in vitro these GIT conditions: a low pH value and a high bile concentration are tested for variable times in order to determinate the survival of the strains under test.

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Targeted screening focused on the application

“Conventional” screening described above consists of in vitro experiments that are mainly important to assess the safety of probiotic strains. However, it is realized that these tests, including identification of strains, safety assessment and characterization of some activities (ability to grow in conditions of low pH and in the presence of bile acids) are not accurate to predict the potential use and the functionality of probiotic strains. So, it’s necessary to expand the knowledge of specific properties of tested strains.

Knowledge about intestinal microbiota, nutrition, immunity, and genetics in health and disease has increased in the past years. This information helps to develop new probiotic strains with disease-specific functions. Probiotics are not a uniform group of microorganisms with health benefits: the efficacy and the properties of each specific strain should be assessed individually rather than as a group of probiotics mainly because different strains exert different effects.

Then, “conventional” screening must necessarily be followed by a screening depending on the application based on the development of platforms for functional characterization, adopting screening criteria for assess if the potential new probiotic is suitable for use in certain areas of consumer health (oncological diseases, cardio-vascular, metabolic, etc.).

Until few years ago, there was the idea that the same strain could be used for different applications, this is not plausible in fact a strain with specific features is usually employed for a specific application. Nowadays in fact is widely supported the thesis that, for the same strain, you can not have an effective use for different applications but, for each application, you must choose the most suitable strain.

This targeted screening is closely related to the benefits of probiotics on human health.

An increasing number of studies have shown that probiotic bacteria are in fact able to positively influence the state of health, thanks to the numerous activities that they carry out, in particular: maintaining a balance in the intestinal microflora, protection against intestinal pathogens and modulation immune response leading to an improvement in allergies food and autoimmune disorders (Figure 2).

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Probiotics

Balance of microflora

Prevention of intestinal infections

Prevention of pathogen adherence

Decrease of allergic reactions

Modulation of immune response

Figure 2: Mechanisms of action of probiotics

Prevention of pathogen adherence

The adhesion of pathogens to the intestinal mucosa appears to be crucial for the beginning of the infection process.

Pathogenic bacteria are in fact able to adhere to the intestinal epithelium, colonize with detrimental effects to epithelial cells.

The microbiota present in the intestine provides a barrier to the adherence of pathogenic bacteria and various mechanisms have been proposed to explain this defensive action of probiotics. Some authors have suggested that the production of organic acids, such as acid lactic and acetic acid by probiotics, especially by some lactobacilli strains, lowers the intestinal pH and inhibits the growth of pathogens. Moreover, the same authors have seen that probiotics stimulate peristalsis that, indirectly, removes pathogens, accelerating the speed of transit in the intestine.

Another mechanism supported by various studies is the ability of probiotics to compete with pathogens in adhering to the intestinal mucosa (Arquès et al., 2015). Some probiotics can inhibit the adhesion of pathogens to their binding sites on the surface of the intestinal membrane by competing for binding.

A study of Gopal et al., 2001, has confirmed that some lactobacilli, like L. rhamnosus DR20, L.

acidophilus HN017 and B. lactis DR10, are able to adhere to intestinal cell lines in vitro and to decrease the colonization and the entry of pathogens into the intestinal mucosa.

17 Prevention of diarrhea and intestinal infections

It’s well known that various strains of lactobacilli can be used to prevent or treat acute diarrhea caused by Escherichia coli, Salmonella or Shigella and this effect is mainly due to the production of bacteriocins by different strains of probiotics used. A study of Davoodabadi et al., 2015 has shown the ability of strains of L. fermentum, L. plantarum and L. paracasei to inhibit the growth of enteropathogenic bacteria including strains of Shigella flexneri, Shigella sonnei, Salmonella enteritidis and Yersinia enterocolitica.

Some authors have used L. rhamnosus GG added to yogurt, to treat diarrhea associated with the treatment by antibiotics as demonstrated by a recent study that evaluated the efficacy of L.

rhamnosus GG in the prevention of antibiotic-associated diarrhea both in children and in adults (Szajewska and Kołodziej, 2015). The administration of antibiotics can lead to an imbalance in the microbiota and a net reduction of the intestinal "beneficial" microflora, the main responsible of the resistance to the colonization of pathogens. The results of this study showed that humans volunteers that received probiotics with the antibiotic showed lower diarrhea compared to individuals that consumed only pasteurized yogurt as control (Siitonen et al., 1990).

Efficacy against diarrheal disease is well documented also with other probiotics strains, such as L. reuteri, L. casei and Saccharomyces boulardii (Huang et al., 2002; Van Niel et al., 2002).

Probiotics are widely used for prevention and treatment of diarrhea more in children than in adults (Guarino et al., 2015): the administration of probiotics, such as B. bifidum, Streptococcus thermophilus and L. rhamnosus, has proved very useful also as prophylaxis to prevent nosocomial diarrhea in children (Szajewska et al., 2001).

The incidence of Crohn's disease and ulcerative colitis collectively called syndrome IBD (inflammatory bowel disease), is constantly increase in industrialized countries. The changes in lifestyle, including a better hygiene and a reduction in the consumption of foods containing bacterial enzymes, may alter the correct microbial balance necessary for the development of a correct intestinal immune system (Shanahan, 2004). This can lead to immune reactions towards the intestinal bacterial flora, and this could be the first step leading to the development of IBD and other disorders related to wrong immune response. Several experimental evidence in fact suggest that both Crohn's disease and ulcerative colitis are caused by hyperactivation of the immune system against intestinal flora, leading to a chronic inflammatory state and a consequent mucosal damage (Shanahan, 2002).

18 The balance between pathogenic and beneficial bacteria in the intestine represents a protection against abnormal immune responses that lead to inflammation.

For this reason a good therapeutic strategy against IBD is a modification of the microflora in favor of probiotics (Fedorak and Madsen, 2004).

Experimental studies carried out ex vivo on intestinal mucosa of patients suffering from Crohn's disease have shown that certain Lactobacillus strains, including L. casei, can induce a decrease of the release of TNF-α (anti-inflammatory signal) by the inflamed mucosa.

Moreover experiments conducted on rats which spontaneously develop colitis, have shown that oral administration of L. reuteri or L. plantarum 299v did significantly decrease the symptoms of the disease (Schultz et al., 2002). Also Torres-Maravilla et al., 2015 demonstrate the potential of a strain of L. sanfranciscensis to treat IBD. Often the symptoms of IBD were treated by using a mixture of probiotics as demonstrated by a study of Yoon et al., 2015 that evaluated the effect of a probiotic mixture composed by L. acidophilus, L. rhamnosus, B. breve, B. lactis, B. longum, and S. thermophilus on the changes in fecal microbiota and IBD symptoms.

They demonstrated that after 4 weeks of administration of this multi-species probiotic mixture the fecal concentration of most probiotic strains increased and the diarrhea-symptoms in IBD patients improved.

Overall, these results show that probiotics act by modulating immune response in IBD thanks to regulatory cytokines, and this suggest an important role for these bacteria for the treatment of IBD.

Stimulation of the immune system and beneficial effects on allergic reactions

Among the various beneficial effects exerted by probiotics very important is their capacity of interaction with the immune system (Jespersen et al., 2015). For this reason it is essential that the immune system recognizes the parts of the microbiota as self and express a tolerance towards them, this tolerance is also possible thanks to the fact that the bacteria of the microbiota do not express virulence factors (Aureli et al., 2011).

The thesis that probiotics may influence immunity thus exerting a beneficial role in the treatment of human diseases is nowadays a topic of great interest.

There are in fact extensive evidence that suggest that lactic acid bacteria are able to stimulate both innate and acquired immune response, the lymphocyte function and the production of antibodies and cytokines (Gill et al., 2000; Pessi et al., 2000).

19 First of all, the ability of probiotics to modulate lymphocyte populations has been demonstrated by some authors who observed an increase in the population of T-helper lymphocytes in rats treated with L. casei (Perdigon et al., 1999).

Furthermore it was seen that some strains of probiotics are able to increase the number of populations of neutrophils and macrophages, as well as to stimulate the activity of the cells natural killer (Gill et al., 2000; Matsuzaki and Chin 2000) which are the first line of defense thanks to their cytotoxic activity exercising against antigens (Ley et al., 2008). Moreover, some strains of lactobacilli induce dendritic cells (DC) maturation that pass through epithelial cells and capture antigens from the lumen.

About the effect of probiotics on the production of antibodies, numerous studies have shown that the treatment with some strains of probiotics is able of enhancing the immune response antigen-specific against natural infections and immunizations, in particular probiotics act by increasing the production at mucosal level of immunoglobulin A (IgA), the first line of defense against pathogenic bacteria and viruses that daily are inhaled and ingested. A more recent study investigated the IgA increase induced by a strain of L. plantarum and demonstrated that this strain increased the IgA level of Peyer's patch (PP) cells, although its mechanism of action is not clear yet. They demonstrated that taking concentrations of 0.03% or 0.3% of L. plantarum powder for 4 weeks, caused an increase of IgA in the small intestine of the mice (Kikuchi et al., 2015). Despite these results indicate the ability of this strain to maintain mucosal immunity, it is necessary to better understand its mechanism of action in order to use this strain in functional food. Another study of Sakai et al., 2014 demonstrated that a strain of L. gasseri stimulates dendritic cells to promote the production of TGF-β, IL-6, and IL-10, all critical for IgA production from B cells activating in this way the signal to produce IgA in the mouse small intestine.

For many years the production of cytokines was associated only with the response against infections and it has been given little attention to the fact that lactic acid bacteria could induce the production of cytokines even in conditions of perfect health. The effect most studied on the immunomodulatory activity of probiotics regards precisely the expression of cytokines, both pro- and anti-inflammatory, at both intestinal and systemic level. Several in vitro studies show an increase in pro-inflammatory cytokines such as IL-12 and tumour necrosis factor-alpha (TNF- α) in the presence of probiotics as evidenced by a study of Nishibayashi et al., 2015 where it

20 was investigated the role of three LAB strains in inducing the production of interleukin-12 from human monocytic cells. Particularly the study was conducted by using a strain of E. faecalis, a strain of L. gasseri and one of B. breve and it was demonstrated that a treatment with RNase A of heat-killed LAB significantly decreased the IL-12 production of human cells demonstrating that the single stranded RNA (ssRNA) of LAB is a strong inducer of IL-12 production from human monocytes. Other authors confirmed the trend of lactobacilli in increasing the production of inflammatory cytokines, finding an increase of IFN-γ and IL-12 in human peripheral blood mononuclear cells treated with L. johnsonii and L. sakei, while the level of IL-10 is not seemed to increase (Haller et al., 2000).

It was also reported an activity of probiotics in inducing the expression of anti-inflammatory cytokines. About that, an in vivo study of Wang et al., 2015 on patients in dialysis evaluated the effect of probiotics on inflammatory markers such as interleukin IL-6 and TNF-α that are elevated in patients with this disease and the impact of the same bacteria on the anti- inflammatory cytokine IL-10. They found that a treatment with a strain of B. bifidum, B.

catenulatum, B. longum and L. plantarum caused a decrease in the levels of serum of pro- inflammatory cytokines TNF-α, IL-5 and IL-6 while levels of serum of IL-10 significantly increased.

These studies confirm the role of the probiotics in the modulation of gene expression associated with the immune system and inflammation. Specific strains of Bifidobacteria and Lactobacilli influence the gene expression of mucins, nuclear factor and interleukins leading to an anti-inflammatory response in the presence of enterocytes in culture (Plaza-Diaz et al., 2014). Moreover, probiotics interact with the surface of antigen-presenting cells in vitro causing the downregulation of pro-inflammatory genes that are linked to inflammatory signaling pathways, while other anti-inflammatory genes are upregulated. These effects of probiotics are widely studied in animal models while information about the impact of probiotics on gene expression in human intestinal cells are very scarce. There is the need of further clinical studies to elucidate the mechanism of action of probiotics both in healthy humans and in patients with chronic diseases. These types of clinical studies are necessary for addressing the influence of these microorganisms in gene expression associated with the immune response to finally better understand the role of probiotics in the prevention and treatment of disease.

21 Then accumulating evidence suggests that probiotic bacteria play an important role in modulating various aspects of immunity and the ability to regulate this type of responses is very useful from the clinical point of view, for example for immunoprophylaxis or, more generally, to increase immunity against pathogens of different nature.

There are several studies that demonstrate a role of probiotics also in autoimmune and allergic disorders.

Food allergies are chronic disorders of growing importance in countries more developed, and they are mainly due to an uncontrolled immune response against specific antigenic determinants present in the environment or in the food.

Considering the anti-inflammatory and immunomodulatory effects exerted by probiotics, it was suggested the use of probiotics as a new strategy for the control of inflammation and allergic reactions, because they are able to favorably alter the microflora of the host and modulate the intestinal immune response.

It is well known that an adequate colonization of the intestine by the bacterial flora in the first years of life is responsible of the proper balance between the Th1 cells and Th2 ones, which provides protection against allergies (Kalliomaki and Isolauri, 2003; Bischoff and Crowe, 2004).

The intestinal microflora can potentially promote anti-allergenic processes, it can in fact stimulates the production of TGF-β and IL-10, resulting in promotion of the oral tolerance (Christensen et al., 2002; McGuirk and Mills, 2002). It was in fact observed a decrease in the inflammatory immune response to food antigens in allergic individuals following treatment with probiotics (Cosenza et al., 2015).

Gut-brain axis

The term probiotic is always associated with the intestinal environment and with the maintenance of its eubiosis. However in recent years has arisen the idea that probiotics can also affect brain functions by helping to improve or prevent disorders such as depression and anxiety. The ability of the microbiota to communicate with the brain is demonstrated by the fact that a decrease in beneficial flora leads to a deterioration of the gastrointestinal and neuroendocrine relationships causing diseases. The collaboration between the brain and the gastrointestinal tract is fundamental for maintaining homeostasis and it is regulated at the level of central and enteric nervous systems (Cryan and O’Mahony, 2011). Perturbation of these

22 systems has as a consequence an alteration in the stress-response and behavior (Rhee et al., 2009). So the enteric microbiome has an impact both on the gut that on the brain and for this reason it was coined the phrase “gut-brain axis”.

It is certain that bacteria in the human gut have the ability to produce molecules with neuroactive functions which could affect the brain. Some typical bacteria of the human GIT are in fact able to produce many neurotransmitters and neuromodulators and, among them, Lactobacillus spp. and Bifidobacterium spp. have been reported to produce γ-aminobutyric acid (GABA) and, especially bacteria of the genus Lactobacillus, have been reported to produce acetylcholine and histamine. These secreted neurotransmitters from bacteria in the intestinal lumen may induce epithelial cells to release molecules that modulate neural signal in the enteric nervous system and consequently the functioning of the brain and the behavior of the host (Wall et al., 2014). It was demonstrated that different strains of Lactobacillus and Bifidobacterium are able to produce GABA when growing in the presence of monosodium glutamate. GABA is a neurotransmitter that in the brain is involved in states of anxiety and depression through the regulation of different physiological processes (Schousboe and Waagepetersen, 2007). Another study of Messaoudi et al., 2011 assessed the effect of the combination of L. helveticus and B. longum on rats and demonstrated that these probiotics reduced anxiety in animals. Although the mechanism of action is not known it has been shown that different probiotic strains are able to modulate the level of inflammatory cytokines (Brenner and Chey, 2009) and to decrease oxidative stress thereby reducing depression and anxiety. A bacterial species able of modulating the state of depression by inhibiting the production of pro-inflammatory cytokines is the B. infantis (Desbonnet et al., 2008).

Several studies have also shown that the use of a combination of probiotics obtained by combining different bacterial genera or different species of the same genera may be more effective than a mono-species supplements (Chapman et al., 2011). However, some strains can compete with others in the execution of their functions so the use of a mixture of strains to obtain a greater effect requires careful verification in the preparation of the mixture itself.

Moreover, these evaluations were usually carried out using animal models, in particular were used germ-free mice or rats. These animals do not have any bacterial contamination, and thus

23 offer the opportunity to study the effect of the complete absence of the intestinal microbiota on behavior. Although these animals are useful for research in the field of neuro- gastroenterology, the results obtained from animal studies can not be extended to human individuals since they are not representative of the real situation of the human population.

Probiotics studies in human are still scarce but available data look promising. For example a study of Benton et al., 2007 found that a treatment of three weeks with a drink based on milk containing L. casei Shirota improved mood scores in individuals with symptoms of depression.

So these data suggest an ability of probiotic strains to modulate some aspects related to brain and behavior, but these results obtained in animal models have to be confirmed in human and there is also the need to understand the molecular and cellular basis of this gut-brain communication mediated by microbiota.

Market of probiotics

The worldwide interest in the field of probiotics is very high, thanks to the many new products launched every year on the market, the interest of researchers in demonstrating the real property and consumers, more and more attentive to health products. The growing awareness of consumers and their attention to new concepts of health and well-being in recent years, favored a significant growth of the global market for probiotics, thereby increasing the level of interest and investment of industries, researchers and producers. Over 500 new probiotics products were introduced in the last decade, although, not everyone receives the same level of success in the market.

Estimates of Euromonitor International at the end of 2014 confirm the positive trend of the markets for vitamins and food supplements in the main countries of Western Europe, with the only exception represented by Germany. For all the other markets, the medium annual growth rate varies from 2.7% in Italy to 3.9% in the UK. Regarding the expenditure per capita, the highest level is reached in Italy with 29,50 €. Italian consumer is followed by the Belgian one with 27,20 €. In addition, in Italy the trend of the category is driven by probiotics and minerals.

Concluding given the rapid evolution of the probiotic market in recent years, companies operating in this field have the main objectives of identify new applications for their products and differentiate the end use to try to further expand and to support the market.

24

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